1. Field of the Invention
The present invention relates to systems and methods for flow cytometry. More specifically, the present invention relates to systems and methods for calculating drop delay in a flow cytometer.
2. Background Art
In the field of flow cytometry, a hydrodynamic focusing technique is used to separate and align analyte particles within a stream. An oscillator perturbs the stream as it is released from a nozzle of the flow cytometer. The perturbation results in droplets containing individual analyte particles. Just prior to breaking off from the stream, the droplets can be electrically charged and an electrical field downstream from the break-off point can be used to deflect and sort the charged particle. As part of this process, it is necessary to determine the system's “drop delay” (i.e., the time between when a particle of interest is identified, to when a droplet containing the particle of interest reaches the charging location (or “break-off point”)). As such, the droplet of interest can be charged without affecting neighboring droplets.
Commercial flow cytometers typically use empirical, iterative, trial-and-error protocols to determine drop delay. For example, fluorescent beads can be run through the flow cytometer while a user (or automated program) cycles through a range of drop delay settings. When the user (or automated program) finds the drop delay setting resulting in the optimal sorting of the fluorescent beads, such drop delay is set as the operational drop delay, and is used for the sample runs. Use of fluorescent beads to determine drop delay, however, is undesirable because fluorescent beads can contaminate the flow cytometer. Further, what are needed are systems and methods to avoid the use of iterative, trial-and-error operations, which are time-consuming and can lack the necessary precision of drop delay calculation.
The systems and methods presented herein act to solve some of the problems seen in the prior art. It is noted that the systems and methods presented herein do not merely automate a previously manual process. The systems and methods presented herein have been developed through painstaking design, testing, and optimization. Challenges in achieving the necessary precision were identified and addressed in the development of these systems and methods. As such, the systems and methods presented herein provide significantly higher accuracy than prior art systems.